In the past, a method for measuring an extent of immunological reaction based upon an intensity of excited fluorescent light is proposed. The excited fluorescent light is obtained by previously fixing antigens or antibodies on a surface of a slab-type optical waveguide, by performing antigen-antibody reaction between antibodies or antigens in a test liquid for measurement and the fixed antigens or antibodies, by performing antigen-antibody reaction between labeled antigens or labeled antibodies labeled with fluorescence dye and the reacted antibodies or antigens, by introducing an exciting light into the slab-type optical waveguide which light propagates in the slab-type optical waveguide in a total reflection manner, so as to generate an evanescent wave component, and by exciting fluorescent dye by the generated evanescent wave component the dye being constrained in the vicinity of the surface of the slab-type optical waveguide. In this case, a so-called end-point method is generally employed which detects an intensity of the fluorescent light when an intensity of the fluorescent light becomes nearly stable and calculates an extent of immunological reaction based upon the detected intensity. Also, a measurement signal obtained by the method is referred to as an end-point signal.
Further, when a measurement is performed by employing the above method, a method is proposed which shortens a required time for measurement by previously performing the antigen-antibody reaction by mixing a test liquid for measurement and labeled antigens or labeled antibodies, i.e., by mixing the test liquid with the labeled antigens prior to performing the immunological measurement (hereinafter, this method is referred to as a 1 step sandwich method).
When an immunity measurement is performed using the former method, antigen-antibody reaction should be performed between the antigens or antibodies fixed on the surface of the slab-type optical waveguide and the antibodies or antigens included in the test liquid for measurement. The liquid for measurement should then be discharged, and the antigen-antibody reaction should be performed between the labeled antigens or labeled antibodies and the reacted antibodies or antigens. Therefore, time for performing the antigen-antibody reaction twice is needed and a time for discharging the test liquid is needed, so that a required measurement time in its entirety is lengthened. Further, when the test liquid is discharged, the test liquid should be discharged up to nearly 100% so that the liquid which includes labeled antigens or labeled antibodies is not diluted by remaining test liquid. When a vessel for housing the test liquid is small in size, a disadvantage arises in that the discharging operation becomes difficult. Of course, when the test liquid is imperfectly discharged, a concentration of the labeled antigens or labeled antibodies is diluted so that a quantity of antigens or antibodies which are constrained in the vicinity of the surface of the optical waveguide by the antigen-antibody reaction is lessened. Therefore, the accuracy of immunological measurement is lowered.
When an immunological measurement is performed using the latter method (1 step sandwich method), an operation for discharging the test liquid is not needed at all, and a required time for an immunological measurement is remarkably shortened by performing the antigen-antibody reaction in parallel to another processing, such as, for example, an immunological measurement processing based upon another test liquid for measurement, because the labeled antigens or labeled antibodies and the test liquid are previously mixed so that the antigen-antibody reaction is previously performed. Of course, a lower accuracy of the immunological measurement due to imperfect discharge of the test liquid is not realized at all.
A disadvantage arises in that the same intensity of fluorescent light is possibly detected when a concentration of antigens or antibodies in a test liquid is high and when a concentration of antigens or antibodies in a test liquid for measurement is low. The disadvantage cannot be ignored because a mistaken measurement result badly affects a human body by taking a condition into consideration which condition that measurement of an extent of immunological reaction is mostly performed on contradiction that medical treatment and the like is performed thereafter.
Occurrence of the above disadvantage is further described in detail.
In the 1 step sandwich method, an antigen-antibody reaction is performed between antigens in a test liquid for measurement and labeled antibodies, by previously mixing a reagent solution which includes labeled antibodies and the test liquid, for example. The labeled antibodies are obtained by labeling antibodies with fluorescent dye. A concentration of antigens in the test liquid is unknown. The mixed liquid is under a condition that the labeled antibodies excessively exist, or the antigens excessively exist, or the labeled antibodies and antigens exist in a nearly balanced condition, depending upon the concentration of the antigens. When the labeled antibodies excessively exist, many of the labeled antibodies exist in the mixed liquid which are not included in the antigen-antibody reaction. When the antigens excessively exist in the mixed liquid, many of the antigens exist in the mixed liquid which are not included in the antigen-antibody reaction. When the labeled antibodies and antigens exist in a nearly balanced condition, the labeled antibodies and the antigens scarcely exist in the mixed liquid which antibodies and which antigens are not included in the antigen-antibody reaction.
The above disadvantage does not occur when the labeled antibodies excessively exist or when the labeled antibodies and antigens exist in a nearly balanced condition. However, when the antigens excessively exist in the mixed liquid, not only the antigens which have already been included in the antigen-antibody reaction with the labeled antibodies, but also the antigens which have not been included in the antigen-antibody reaction with the labeled antibodies, are included in the antigen-antibody reaction with the antibodies which are fixed on the surface of the slab-type optical waveguide. When a quantity of the latter antigen-antibody reaction increased, a quantity of the labeled antibodies which are constrained in the vicinity of the surface of the slab-type optical waveguide is lessened. Therefore, the same intensity of fluorescent light is obtained in cases where the concentration of antigens in the test liquid is low and where the concentration of antigens in the test liquid is high. Consequently, it becomes impossible to determine a concentration of antigens in a test liquid for measurement uniquely.
To resolve such a disadvantage, it is necessary that another measurement be performed based upon the 1 step sandwich method again, after the test liquid for measurement is diluted, to judge whether already obtained measurement result is correct. Specifically, when signal values of 89 and 88 arbitrary units are obtained based upon intensities of fluorescent light in cases where the test liquids for measurement is not diluted, which liquids have concentrations of .beta.2-microglobulin of 10 .mu.g/ml and 300 .mu.g/ml, respectively, signal values of 11 and 131 respectively are obtained based upon intensities of fluorescent light when the test liquids for measurement are diluted by ten times. Therefore, concentrations of .beta.2-microglobulin are uniquely determined to be 10 .mu.g/ml and 300 .mu.g/ml, respectively. As a result, an extent of immunological reaction is fairly accurately measured. But, new disadvantages arise in that a required time for measurement is remarkably lengthened for the measurement's entirety and that a required quantity of labeled antibodies for measurement is increased. Of course, a required number of optical waveguides on which antibodies are previously fixed also is increased. Further, a disadvantage arises in that the accuracy in measurement of a degree of the antigen-antibody reaction is lowered because a measurement error is generated due to differences between measurement timings and between optical waveguides on which the antibodies are fixed, and the like.